Peixuan Guo - US grants

Guo, P. MCB 9723923 Technical A virus-encoded RNA (pRNA) is required for the packaging of phage phi29 dsDNA into a preformed procapsid during maturation. Packing of DNA also requires the DNA packaging protein gp16, ATP and Mg++. A highly sensitive in vitro assembly system has been established to study phi29 DNA package. With the aid of pRNA and cloned gene products, DNA can be packaged in vitro with up to 90% efficiency and up to 108 plaque forming units per ml of infectious virions can be assembled without background. In the presence of MG++, 6 pRNAs bind to the protal vertex of procapsids (the site of DNA entry) and are released after DNA packaging is completed. It is known that the pRNA has two functional domains, one for procapsid binding and one for an unknown but essential role. The requirement of the pRNA for ATPase activity of the DNA packing protein suggests a role for the pRNA in the energetics of package. The hypothesis being tested is that the pRNA, acting as part of a DNA packing ATPase, undergoes a series of conformational changes during DNA packaging. The conformation of the pRNA in the presence of ATP is investigated by chemical and nuclease probing and crosslinking. Non technical The study of pRNA conformational changes as it relates to phage phi29 DNA packaging is to provide basic information regarding viral assembly, RNA structure, bioenergetics, and molecular motion. As the involvement of the phi29 pRNA in DNA translocation is unique among known RNA functions, investigation of the structure and function of this molecule has broad significance to the mechanisms of RNA/ATP, RNA/Mg++, RNA/protein, RNA/DNA, and RNA/RBNA interactions. Additionally, the study of the phi29 DNA translocation process may have relevance to other DNA translocation enzymes, such as helicase, as both processes can utilize ring-shaped structures with six-fold symmetry.

The study of viral genome encapsidation has attracted increasing attention recently as it could reveal potential targets for anti-viral therapy and have relevance to other biochemical processes, such as molecular motion, bioengergetics, and macromolecular interactions. Extensive investigation on DNA packaging of the double-stranded DNA viruses documents certain common features in this step of the viral life cycle. Bacteriophage phi 29 encodes a small RNA (pRNA) that is indispensable for genomic DNA packaging. Recent work has revealed that several antisense oligonucleotides to pRNA completely block virion assembly in vitro, and that mutant pRNAs completely block virion assembly in vivo. The long term objective of this study is to determine the largely unknown role of the pRNA in phi 29 DNA packaging, to revela the basis of RNA/protein, RNA/DNA and RNA/RNA interactions, as well as to elucidate the mechanism of the RNA- involved, energy-driven DNA translocation into the procapsid. In this proposal, specific bases or gions of the pRNA will be labeled by the use of circularly permuted pRNAs and bipartite pRNAs. Intramolecular interactions of the aforementioned labeled pRNA as well as its interactions with other components involved in DNA packaging will be investigated by means of crosslinking. Intramolecular crosslinking will also be used to study conformational changes of the pRNA in the presence of various components of the DAN packaginh machine. The tertiary structure of the pRNA will be determined based on the previous solving of the pRNA secondary structure.

virus; growth factor; genetics; biomedical resource; bacteria;

Migration or translocation of macromolecules, such as proteins or nucleic acids, through barriers or cell membrane is a common process in biological system. One of the most complex and intricate translocation processes is viral DNA encapsidation. All linear dsDNA viruses, including hepesviruses, poxviruses, adenoviruses, and the ds-DNA bacteriophages package their genomic DNA into a preformed protein shell (procapsid). ATP is consumed to provide energy to condense the lengthy genomic DNA into the limited space inside procapsids with remarkable velocity. Phage phi29 requires a viral-encoded 120-base RNA (pRNA) to package its genomic DNA. This pRNA contains two single-stranded loops that are responsible for inter-RNA interactions. Through this loop/loop interaction, six pRNAs form a hexameric ring to gear the DNA packaging machine. We hypothesize that the pRNA possesses at least two functional conformations. Alternating between contraction and relaxation, driven by ATP hydrolysis, of each member of the pRNA hexagon could help rotate the DNA transportation machinery. Our long term objective is to elucidate how this DNA transportation motor works, thus providing fundamental information concerning bioenergetics as well as the mechanism of the interactions among protein, DNA and RNA. The short term objective of this proposal is to investigate the conditions for the formation of pRNA dimers or hexamers, and to construct hexagonal arrays composed of hexameric pRNA complex for future structural analysis.

DESCRIPTION (Adapted from abstract): The in vitro assembly of phage phi29 provides a system for developing methods to determine the stoichiometry of components in complex systems. Assembly from purified constituents generates 108 virions per ml, so this is an especially sensitive system for examining the effects of specific mutants. Two complementary methods have been used to determine the stoichiometry of an RNA oligomer that is required for DNA packaging of the phi29 capsid. In the first method empirical data are compared to theoretical curves generated by a binomial expression, assuming certain stoichiometries. In the second method, the dilution curves (log/log plots of component concentration versus yield of product) of components of known stoichiometry are compared to that of the unknown. The objective of the current proposal is to use these same methods to determine the stoichiometry of two additional constituents of phi29, gp16 and gp12. In the case of the former protein the analysis is complicated by the fact that gp16 acts catalytically in the DNA packaging step. This provides an opportunity to examine the influence of reusability on these two methods for determining stoichiometry. It is hoped that the types of analysis developed here will be used to study other complex biological systems.

DESCRIPTION (provided by applicant): Nanotechniques involve the creation, characterization, and modification of organized nanomaterials to serve as building blocks for the construction of large-scale devices and systems. Living systems contain a wide variety of nanomachines and highly ordered structures, including motors, arrays, pumps, membrane cores, and valves. The novelty and ingenious design of bacterial virus phi29 DNA packaging motor, the strongest biomotor studied to date, have inspired the synthesis of this motor and its components as biomemitics for nanodevices. This 30 nm nanomotor uses six ATP-binding pRNA molecules to gear the motor. An imitative motor has been successfully constructed using purified recombinant proteins and artificially synthesized RNA. It can be turned on and off at will. The formation of ordered structural arrays of the motor complex and its components, the retention of motor function after 3'-end extension of the pRNA, and the ease with which pRNA dimers and trimers can be manipulated combine to make the RNA-containing motor a viable option as mechanical parts in nanotechnology. The long-term objective is to utilize this synthesized motor, together with arrays of its components, as parts in nanodevices, with several major applications: 1) To use this artificial motor as parts for nanodevices, such as apparatuses for in vivo drug delivery. 2) To use the ordered arrays of motor components as building facades for large-scale supramolecular structures to serve as molecular sieves, chips for the diagnosis of diseases, or as ultrahigh density data storage systems. The presence of six pRNA subunits in the hexameric building blocks will allow for the construction of arrays with multiple functions, for example to allow for the separate identification of multiple pathogens. 3) To develop this nanomotor into a DNA-sequencing apparatus, since the DNA-packaging process involves movement of the DNA through a 3.6-nm pore surrounded by six RNA that can be modified to accept chemical signals. Though all of these long-term applications are becoming more and more realizable, practical nanotechnology is still in its infancy. It would be unrealistic to propose applying the phi29 motor system directly to nanodevices in only three or four years. Thus, the short-term objective of this proposal is to construct pRNA or protein arrays that will serve as templates for the construction of patterned supramolecular structures, and to find the best routes through which to link nanotubes and nanogolds as well as to attach biological moieties and chemical groups to the motor or the constructed arrays, pRNA dimers, trimers and hexamers will be used for array construction. Chief among early concerns will be avoiding disruption of the assembly and functioning of the motor and/or arrays due to the incorporation of foreign components. This phase of the study will pave the way toward direct and practical technological applications of the motor and its arrays in nanodevices.

Migration or translocation of proteins or nucleic acids through barriers or cell membranes is a common process in biological systems. One of the most complex and intricate translocation processes is viral genomic DNA packaging. A strikingly common feature in the maturation of linear dsDNA viruses, including herpes viruses, adenoviruses, and the ds-DNA bacteriophages, is that the lengthy genome is translocated with remarkable velocity into a limited space within a preformed protein shell and packaged into near-crystalline density. A DNA-packaging motor accomplishes this energetically unfavorable motion reaction. The bacterial virus phi29 DNA packaging motor contains six pRNA molecules, which bind ATP and form a hexamer as a vital part of the motor. This pRNA contains two function domains, one domain is for binding to the protein component of the motor, and the other is the DNA-packaging domain, but the detail structure and function of this domain is unknown. We hypothesize that the alternation between contraction and relaxation, driven by ATP hydrolysis, of each member of the hexameric RNA ring could rotate the DNA transportation machinery. Our long-term objective is to elucidate the biochemical and biophysical mechanism of this RNA-containing motor, thus providing fundamental information concerning antiviral therapy, bioenergy conversion, macromolecular interactions, nanomotors as parts for nanodevices, and the general mechanism(s) of biomotors. For instance, the motor components of cytomegalovirus have been used as targets for antiviral drug design. The short-term objective of this renewed grant is to continue the study on the structure and function of the hexamer pRNA, focusing on the structure and function of the DNA packaging domain. The stoichiometry of pRNA on the motor before, during and after DNA packaging will be confirmed. ATP-binding and possible ATPase activity of pRNA will be further investigated by crosslinking and ATPase assays under various conditions. Photoaffinity crosslinking will be used to determine the target, if any, the DNA-packaging domain binds or is adjacent to. The specific pRNA/protein interactions will be probed to refine our understanding of the 3D structure of the motor complex. Probing for such precise interactions involves the detection of both the specific nucleotides of pRNA that bind to the connector and the specific amino acids of the connector protein that contacts the pRNA. Fluorescent beads or nanotubes will be used to label the components to allow direct observation of the direction of motion.

Innovative biological machines, such as nanomotors and membrane channels, inspire the development of biomimetic devices that can provide the exquisite control of cellular processes for disease diagnostics and treatment. The phi29 is the strongest biomotor constructed to date. The motor is switchable, underscoring its highest efficiency in DNA insertion compared to all other in vitro viral packaging motors, making the motor a viable option as a mechanical component in nanotechnology applications. The long-term goal of the proposed Nanomotor Drug Delivery Center is to create biologically compatible membranes and arrays with embedded phi29 DMA-packaging motors for applications in medicine. This will be accomplished by focusing on three key areas of study: reverse engineering the phi29 motor;incorporating the active nanomotor into lipid bilayers;and developing active nanomotor arrays that enable drug delivery and diagnostics. The short-term goal of this center is to create liposomes and array structures with embedded phi29 DNA- packaging motors for both passive and active transport of DNA and drugs. The Center will develop hybrid systems that combine the best features of the biological nanomotor and synthetic delivery systems that have already achieved clinical acceptance. Initial efforts will provide engineering specifications for the structure and working mechanism of the phi29 nanomotorthat will enable us to produce engineered, hybrid, synthetic and functional nanomotors. Incorporation of active phi29 nanomotors in liposomes will be a major thrust that will enable nanoscale control for therapeutic approaches that have already gained acceptance in the clinic but still provide only blunt instruments for therapy of complex conditions such as cancer and infectious diseases. Additional team efforts will exploit our understanding of the reengineered membrane-addapted motor to create prototype nanomechanical devices for diagnostic applications, targeted delivery of therapeutics and for various nanomachines with application in medicine. These applications and the engineering principles underlying the physical action, energy transduction and transmembrane transport capabilities of the membrane adapted motors will be elucidated by our interdisciplinary team including physicists, chemists, engineers, mathematicians, physicians, and molecular biologists.

DESCRIPTION (provided by investigator): Nanotechniques involve the creation, characterization, and modification of organized nanomaterials to serve as building blocks for the construction of submicro or nanoscale devices and systems with applications in technology and medicine. Living systems contain a wide variety of ordered macromolecular structures and powerful bionanomachines. The ingenious, novel design of the bacteriophage phi29 DNA packaging motor and its constituents has inspired the artificial synthesis and assembly of phi29 biomimetic motor and its components. The 30-nm nanomotor was geared by six copies of ATP-binding packaging RNAs (pRNAs). The structural versatility of pRNA coupled with its ability to form dimers, trimers, hexamers and patterned superstructures via interlocking loop interactions make it a promising tool for nanomachine fabrication, pathogen detection and drug/gene delivery. The low-resolution global structures of the pRNA and its dimer, trimer and hexamer have been probed by photoaffinity cross linking, chemical modification interference, compensatory modification, Cryo-AFM and 3D computer modeling. The long-term objective is to introduce exogenous components, nano-materials, modules, moieties, drugs, and other therapeutic reagents into the motor and develop it for various applications in nanotechnology and medicine. During the first four years of this grant, we have demonstrated that pRNA can be used as a building block for bottom-up assembly of polyvalent nanoparticles to deliver siRNA, ribozymes and other therapeutics to specific cells. A single molecule fluorescence dual-view imaging system was designed and constructed to count the number of pRNA copies within the constructed pRNA nanoparticles. The short-term objective of this application is to utilize prostate cancer as a model system to determine the feasibility of using the phi29 pRNA motor for targeted delivery of siRNA or other therapeutic molecules and treatment of cancerous diseases. Specifically, the novel RNA nanotechnology approach previously developed for the bottom-up assembly of multimeric pRNA nanoparticles will be exploited to construct polyvalent pRNA vehicles carrying an RNA aptamer to specifically bind to prostate surface antigens and a therapeutic siRNA targeting an anti-apoptosis factor for the prostate cancer treatment. Methods for large scale production of pRNA nanoparticles and construction of stable pRNA therapeutic nanoparticles resistant to RNase digestion in vivo will be developed. Approaches designed to combine therapy with the simultaneous detection of the therapeutic effect will be developed by exploiting the polyvalent nature of the pRNA nanoparticles. The efficacy in prostate cancer therapy, the biodistribution, pharmacokinetics, toxicity, off-target effects as well as the double stranded RNA induced interference will be evaluate in both prostate cancer cells and mice models. PUBLIC HEALTH RELEVANCE: The novel RNA nanotechnology of bottom-up assembly for phi29 motor pRNA nanoparticles will be used to construct polyvalent pRNA nanoparticles for siRNA delivery in prostate cancer therapy. Prostate cancer will be used as a model system to determine the feasibility of using the phi29 motor pRNA nanoparticles for the targeted delivery of siRNA or other therapeutic molecules and treatment of cancerous diseases.

DESCRIPTION (provided by applicant): This project is to organize an interdisciplinary team to work on RNA nanotechnology by constructing polyvalent RNA nanoparticles for specific targeting and delivery to cancer cells. RNA molecules can down-regulate specific gene expression in cancer cells. RNA is particularly attractive as a building block for bottom-up assembly in nanotechnology and nanomedicine. RNA can be manipulated as easily as DNA, but possesses the versatility in structure and function similar to that of proteins. RNA contains single-stranded stem-loops for intra- or inter-RNA interactions which can serve as mounting dovetails, providing advantages over external linking dowels in nanomachine assembly. This CNPP operation comprises of three major functions: the fundamental studies on therapeutic RNA nanoparticle construction; the conjugation and incorporation of therapeutic and targeting moieties to RNA nanoparticles; and the specific delivery of therapeutics to cancer cells. The team includes basic scientists with strong backgrounds in biomedical engineering, chemistry, and RNA and DNA nanotechnology; cancer biologists with extensive experience in ribozyme and siRNA delivery; and pharmaceutical cooperation focusing on RNA therapeutics with expertise in animal trials. The team will elucidate the principles underlying the RNA/RNA interactions in RNA nanoparticle assembly using phi29 motor pRNA system and RNA junction motifs to build polyvalent RNA oligomers containing aptamer, siRNA, ribozyme, ligand, imaging markers or drugs for cancer cell recognition and gene silencing. A new methodology of SELEX will be developed to screen for stable and high affinity RNA aptamers that target and enter cancer cells specifically. Simultaneous delivery and detection will be designed, combining therapy and detection of subsequent therapeutic effects on apoptosis. Approaches of crossover, chemical modification, and cross-linking will be applied to make RNA nanoparticles stable in vivo. Novel fermentation approaches and industry scale production methods will be developed to produce large-scale stable RNA for clinical applications. Animal trials on pharmacokinetics, bio-distribution, toxicity, gene silencing effects, and cancer cell killing will be carried out on animal models of lung cancer, ovarian cancer, liver cancer, and leukemia. RELEVANCE (See instructions): Cancer is a current and long term health care crisis and treatments necessitate the investigation of specific delivery to target cells. This application proposes to fabricate RNA nanoparticles to incorporate therapeutic SiRNA, aptamers, and ribosomes to accomplish targeted delivery. Therapeutic constituents will be selected for the treatment of lung cancer, ovarian cancer, liver cancer, and leukemia.

DESCRIPTION (provided by applicant): A wide variety of highly-ordered structures of macromolecules and organized nanomachines exist in biological systems for transporting polynucleic acids and polypeptides. The ingenious design of the bacteriophage phi29 DNA packaging motor is an intriguing element in this category. All linear dsDNA viruses package their genome into a preformed procapsid via an ATP-driving motor involving two nonstructural enzymes or ATPases. The central component of the phi29 motor is the portal connector composed of twelve copies of the protein gp10, which form a dodecamer channel that acts as a pathway for the translocation of double-stranded DNA. With a diameter of 3.6 nm at its narrowest end, it is one of the largest among typical channel proteins that have been incorporated in lipid membranes. Explicit engineering and pore modifications of the phi29 connector are possible due to its available crystal structure, thus inspiring its biomedical applications. The short-term objective of this application is to demonstrate the utility of the lipid-embedded channel of the phi29 DNA packaging motor as a highly sensitive device for characterizing biopolymers at single molecule resolution using electrophysiological assays. The connector will first be reengineered for added functionality and various approaches to modulate the conductance of the connector will be systematically investigated. The dimensions of the pore and charge distributions will then be altered by mutagenesis for specific pore modifications in a controlled fashion to ensure optimal sensing. Finally, the channel will be designed to act as a stochastic biosensor and functionalized with modifying groups to recognize nucleotide bases of double stranded DNA for potential sequencing applications. The long-term objective is to develop the channel into a robust sensing device for detecting pathogens and chemicals at extremely low concentrations for disease diagnosis and environmental monitoring, and to utilize it as a high throughput nanopore-based apparatus for the next generation sequencing of double stranded DNA. The channel can further be used as a tool to assay the structure and function of biological molecules and complexes as well as future avenues for therapeutic DNA loading, packaging, and gene delivery to specific cells for the treatment of cancers, viral infections, and genetics diseases. PUBLIC HEALTH RELEVANCE: The membrane adapted bacteriophage phi29 portal motor nanopore protein will be modified, characterized and developed for highly sensitive detection of pathogens and chemicals at extremely low concentrations as well as for characterizing double-stranded DNA at the single molecule level. The channel can further be developed as a tool for therapeutic DNA packaging and gene delivery to specific cells for the treatment of cancers, viral infections, and genetics diseases.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop exosome-like nanoparticles as a delivery vehicle for targetable delivery of extracellular RNA (exRNA) primarily focusing on miRNAs which modulate gene expression and induce immune responses upon delivery in mouse tumor models. Development of exRNA-based therapeutics must overcome challenges including issues of delivery, potential off-target effects, safety, toxicity, the cost of large scale production, elimination of potential biohazards to the environmen and tissue specificity. Unlike the situation with artificially synthesized nanoparticles or liposomes, naturally derived nanoparticle-size exosomes are released from many different types of cells. miRNAs and RNAs can be encapsulated in naturally derived exosomes and the RNAs are stable, and a number of miRNAs can be naturally encapsulated simultaneously in these exosomes. Recently, data published by ours and other groups have demonstrated that mammalian exosomes can deliver poorly soluble agents, chemotherapeutic drugs, and siRNA in vivo. A limitation of this mode is the need for large-scale production of exosomes, and potential biosafety. Our preliminary data show that exosome-like nanoparticles can be isolated in large quantities from the tissue of edible plants, including grapes. We have demonstrated that exosome-like nanoparticles from grapes are composed of small RNAs, proteins, and lipids. An edible plant-derived nano-vector (EPNV) assembled from grape exosome-like derived lipids is capable of encapsulating siRNAs. Co-delivery of folic acid, with the chemotherapy drug paclitaxel by EPNVs results in an increased efficiency in tumor tissue targeting. Reducing the size of EPNVs by passing them through an appropriate pore size filter significantly enhances EPNV translocation to the brain and oral administration of the EPNVs results in their migration to the liver. Based on these preliminary data and the current literature, we propose to: (1) Determine whether an EPNV can deliver therapeutic miR17 via oral administration to target metastatic colon tumor in the liver, subsequently inducing NK cell activation to kill metastatic tumor cells; (2) Determine whether miR155 encapsulated in EPNV promotes differentiation of myeloid derived suppressor cells (MDSCs) into mature dendritic cells in mouse breast/brain tumor models; (3) Determine whether a therapeutic dose of miRNA17 or miRNA155 delivered by EPNV induces no side-effects in mouse tumor models; and (4) Determine whether EPNV can be produced in large scale amounts economically for use in clinical settings. Demonstration of using a EPNV-based delivery system loaded with a desired therapeutic extracellular RNA (exRNA) including microRNA to target to inflammatory cells and tumor cells in vivo would not only be a significant step forward in the treatment of cancer, but also provide a powerful tool for identifying the role and effects of each small RNA encapsulated in resident or circulating microvesicles on the recipient cells. We will accomplish this in our study by knock-out and knock-in strategies.

PROJECT SUMMARY All linear double-stranded (ds) DNA viruses package their genome into a preformed procapsid via an ATP-driving motor. The central component of the phi29 DNA-packaging motor is the portal connector composed of twelve copies of the protein gp10, which form a dodecamer channel that acts as a pathway for the translocation of dsDNA. With a diameter of 3.6 nm at its narrowest end, it is one of the largest among typical channel proteins that have been incorporated into lipid membranes. Explicit engineering and pore modifications of the phi29 connector are possible due to its available crystal structure, thus inspiring the use of such a system as a module in biomedical applications or nanodevices. The short-term objective of this proposal is to demonstrate the utility of the lipid-embedded channel of the phi29 DNA packaging motor as a highly sensitive device for capture and fingerprinting of chemicals and biopolymers in real time at single molecule resolution using electrophysiological assays. The connector will be reengineered for added functionality and various approaches will be undertaken to modulate the dimensions of the pore. Alteration of the charge distributions will be made by mutagenesis in a controlled fashion to ensure optimal sensing. The channel will be designed to act as a stochastic biosensor and functionalized with recognition groups to bind various analytes of interest. Methods to increase the stability and lifetime of the lipid/connector complex will be developed to facilitate high throughput electrophysiological measurement of a single pore with provisions for rapid and efficient sample and buffer exchange. The long-term objective is to develop the channel into a robust sensing device for detecting biomolecules or chemicals at extremely low concentrations in the presence of many contaminants for a wide range of applications in biotechnology, earlier disease diagnosis, environmental surveillance, custom quarantine, drug testing and national security.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop exosome-like nanoparticles as a delivery vehicle for targetable delivery of extracellular RNA (exRNA) primarily focusing on miRNAs which modulate gene expression and induce immune responses upon delivery in mouse tumor models. Development of exRNA-based therapeutics must overcome challenges including issues of delivery, potential off-target effects, safety, toxicity, the cost of large scale production, elimination of potential biohazards to the environmen and tissue specificity. Unlike the situation with artificially synthesized nanoparticles or liposomes, naturally derived nanoparticle-size exosomes are released from many different types of cells. miRNAs and RNAs can be encapsulated in naturally derived exosomes and the RNAs are stable, and a number of miRNAs can be naturally encapsulated simultaneously in these exosomes. Recently, data published by ours and other groups have demonstrated that mammalian exosomes can deliver poorly soluble agents, chemotherapeutic drugs, and siRNA in vivo. A limitation of this mode is the need for large-scale production of exosomes, and potential biosafety. Our preliminary data show that exosome-like nanoparticles can be isolated in large quantities from the tissue of edible plants, including grapes. We have demonstrated that exosome-like nanoparticles from grapes are composed of small RNAs, proteins, and lipids. An edible plant-derived nano-vector (EPNV) assembled from grape exosome-like derived lipids is capable of encapsulating siRNAs. Co-delivery of folic acid, with the chemotherapy drug paclitaxel by EPNVs results in an increased efficiency in tumor tissue targeting. Reducing the size of EPNVs by passing them through an appropriate pore size filter significantly enhances EPNV translocation to the brain and oral administration of the EPNVs results in their migration to the liver. Based on these preliminary data and the current literature, we propose to: (1) Determine whether an EPNV can deliver therapeutic miR17 via oral administration to target metastatic colon tumor in the liver, subsequently inducing NK cell activation to kill metastatic tumor cells; (2) Determine whether miR155 encapsulated in EPNV promotes differentiation of myeloid derived suppressor cells (MDSCs) into mature dendritic cells in mouse breast/brain tumor models; (3) Determine whether a therapeutic dose of miRNA17 or miRNA155 delivered by EPNV induces no side-effects in mouse tumor models; and (4) Determine whether EPNV can be produced in large scale amounts economically for use in clinical settings. Demonstration of using a EPNV-based delivery system loaded with a desired therapeutic extracellular RNA (exRNA) including microRNA to target to inflammatory cells and tumor cells in vivo would not only be a significant step forward in the treatment of cancer, but also provide a powerful tool for identifying the role and effects of each small RNA encapsulated in resident or circulating microvesicles on the recipient cells. We will accomplish this in our study by knock-out and knock-in strategies.

DESCRIPTION (provided by applicant): Development of image-guided drug delivery systems for real-time monitoring, quantifying and validating the delivery and therapeutic action is very important for both research and clinical applications. Various multifunctional nanoparticles of different materials have been investigated over the last decade as prospective imaging and therapeutic platforms; however, effective strategies to quantitatively evaluate delivery of therapeutic payloads to tumors and metastatic cancer cells in vivo are challenging due to low efficiency in specific cancer targeting and non-specific trapping in vital organs such as, liver an lungs. Our goal is to adopt an innovative RNA nanotechnology approach to construct ultrastable multifunctional RNA Beacons and RNA Dendrimers as image-guided agents for quantitative real-time assessment of tumor and metastatic cancer targeted therapeutic delivery, distribution, uptake and response. We have shown that our RNA nanoparticles are non-toxic, non-immunogenic and capable of penetrating across heterogeneous biological barriers to deliver high doses of therapeutics specifically to solid tumors and metastatic cells in mice with little accumulation in normal organs. This provides an ideal platform for non-invasive imaging of cancer therapeutics in vivo. Our multifunctional RNA nanoparticles will be designed to harbor: (1) imaging modules: NIR fluorophores for in vivo fluorescence imaging; radionuclides for PET/SPECT imaging; and gadolinium contrast agents for MRI; (2) targeting modules: such as RNA aptamers or chemical ligands for binding to cancer specific cell surface receptors resulting in internalization of RNA nanoparticles into cancer cells; and (3) therapeutic modules: siRNAs and anti-miRNA to silence the expression of oncogenic genes. The multifunctional RNA constructs will then be evaluated in our well established colon cancer animal models. We will employ multimodal imaging techniques (NIRF, PET/SPECT and/or MRI) for monitoring the therapeutic delivery process in real-time at various spatial and temporal resolution scales. This approach is based on the consideration that each of the three imaging modalities has advantages, and an integrated approach will lead to synergistic benefits with regards to image-guided therapy.

? DESCRIPTION (provided by applicant): Although progress has been made in survival of patients with earlier stage colorectal cancer (CRC), only minimal improvement has been noted in patients with systemic metastases (Stage IV disease). Current chemotherapeutic agents, while highly effective at killing CRC cells, are limited by their systemic toxicity. If advances ar to be made in the survival of cancer patients, highly innovative strategies are required for more targeted delivery of anti-cancer agents directly to CRC metastases. Our ultimate translational goal is to develop a highly effective and less toxic approach to specifically deliver anti-cancer agents to CRC metastases. To achieve this goal, we have assembled a multidisciplinary and highly collaborative team who are at the forefront of molecular signaling pathways in CRC, CRC treatment modalities, medicinal chemistry, novel nanoparticle synthesis and delivery systems. Over the last two years, we have made significant progress to achieve our goals: (i) using a novel three-way junction (3WJ) motif, we have constructed thermodynamically and chemically stable three-branched RNA nanoparticles with an aptamer against receptors that can deliver a small molecule inhibitor or chemotherapeutic agent specifically to CRC metastases in the liver, (ii) we have constructed a variety of RNA nanoparticles using the pRNA-3WJ motif as a scaffold and have demonstrated in critical experiments that the resulting RNA constructs retained their folding and independent functionalities for specific cell binding, cell entry and cancer targeting, both in vitro and in vivo, (iii) we have shown that the RNA nanoparticles remain intact after systemic injection into mice and strongly bind to tumors with little accumulation in normal organs or tissues; these RNA constructs are non- toxic, non-immunogenic, and display favorable pharmacological profiles in mice, and (iv) we have demonstrated localized in vivo delivery of pRNA-3WJ to CRC xenografts and liver metastases. Thus, the central hypothesis of our proposal is that CRC receptor-specific delivery of chemotherapeutic agents using our pRNA-3WJ nanoparticles will provide a safe, effective strategy to selectively target and inhibit CRC metastasis. To address our hypothesis, we have designed experiments with the following Specific Aims: 1) to construct pRNA-3WJ nanoparticles coupled with anti-cancer agents and analyze their stability, cellular uptake and anti-proliferative effects in vitro; 2) to determine te pharmacokinetics, stability, safety and drug delivery of pRNA-drug conjugates in vivo; and 3) to evaluate the selective delivery and in vivo anti-tumor effect of pRNA- 3WJ nanoparticles coupled with anti-cancer agents. In summary, our enthusiasm for our current proposal is driven not only by its inherent scientific importance, but also by its translational potential, clinical impact, and the possibility to provide a more effective and less toxic delivery system targeting CRC metastases.

PROJECT SUMMARY RNA nanotechnology has progressed rapidly during the past several years. This nanotechnology includes the integration of multiple functional modules into one nanoparticle, of which the scaffolds, ligands, therapeutics, and regulators can be composed mainly or exclusively of RNA. We have constructed RNA nanoparticles of diverse size, shape, and stoichiometry displaying high chemical and thermodynamic stability and demonstrated their ability to harbor different functional groups, such as siRNA, miRNA, ribozyme, drug, and cancer targeting RNA aptamer. All functional modules retain their authentic folding and independent functionalities for specific cell binding, gene silencing, and cancer targeting in vivo. Upon systemic injection in tumor bearing mice, RNA nanoparticles bind to xenograft and metastatic tumors specifically and strongly with little to no accumulation in healthy vital organs and tissues 3-4 hours post-administration. The RNA nanoparticles are non-toxic and display favorable biodistribution and pharmacokinetic profiles. Our long-term goal is to promote RNA nanoparticles as a new generation of drug for the treatment of cancers in the clinic. The short-term goal of this project is to characterize the behavior of RNA nanoparticles in vitro and in vivo, with an aim to improve the efficiency for specific cell targeting, internalization and intracellular trafficking, favorable biodistribution without entrapment in liver, endosome escape, and tumor regression. These studies are based on three central hypotheses: (1) intracellular trafficking pathways and endosome escape are critical for effective cancer therapy; (2) biodistribution and pharmacological profiles of RNA nanoparticles are shape and size dependent; and, (3) immune responses elicited by RNA nanoparticles are highly dependent on RNA sequence, chemical modifications, size, shape, and stoichiometry. To address our goals, we will (1) systemically dissect the intracellular pathways taken by RNA nanoparticles and enhance their endosome escape capabilities; (2) inspect the pharmacokinetics (PK); pharmacodynamics (PD); and biodistribution of RNA nanoparticles with the goal of enhancing cancer targeting with minimal accumulation in healthy organs; and, (3) evaluate the immune responses of RNA nanoparticles to minimize non-specific side effects, as well as develop methods to stimulate the immune system by incorporating immuno-stimulatory modules to RNA nanoparticles for cancer immunotherapy. Upon completion of these pre-clinical studies, we will have identified several RNA nanoparticles with optimized shape, size, and stoichiometry displaying favorable safety profiles and high therapeutic efficacy to comply with FDA Investigational New Drug guidelines for initiating clinical trials.

? DESCRIPTION (provided by applicant): Breast cancer is generally asymptomatic at early stages of the disease. Current state of the art screening tools such as mammography and breast examination miss early breast cancers due to the absence of mass lesions, are ineffective in detecting cancer in young women, and require invasive biopsy. New methods are needed to complement current methods for the detection and prognosis of precancerous and cancerous breast lesions while they are small and more easily treated. The analysis of the secreted glycoantigens, protein antigens and non-coding microRNAs (miRNA) in nipple aspirate fluid represents a very promising approach able to detect cancer-related biomarkers directly in body fluids mirroring the breast tissue-specific tumor microenvironment. Our long term goal is to develop a simple, sensitive and robust detection platform for diagnosing diseases at an asymptomatic early stage. Our diagnostic platform is based on the elegant and elaborate protein channel of a biological motor. We have inserted the motor channel into a lipid bilayer and the resulting system exhibits robust properties and generates extremely sensitive, stable and reproducible conductance signatures when analytes interact with the channel. We further demonstrated its utility as a highly sensitive device for detection of cancer-associated proteins i presence of many contaminants at single molecule level. The short-term goal of this proposal is to develop this motor channel as a nanopore for high-throughput single molecule detection of glyco-antigen, protein antigen and miRNA biomarkers extracted from breast cancer patient nipple aspirate fluid. The platform consists of: a membrane-embedded motor channel serving as a highly sensitive nanopore; programmable probes for capturing breast cancer associated antigens; and ultra-sensitive electrophysiological setup for multiplexed high-throughput single molecule detection. Incorporating our device into routine practice will enable physicians to monitor cellular changes within breast ducts earlier, before they develop into larger, potentially cancerous lesions.